RNA interference (RNAi) is a relatively new technology that enables the knockdown of expression of pathologic genes with short interfering RNAs (siRNAs). The limiting factor in the development of new RNAi medicines is delivery. The goal of the field is "Intravenous RNAi," which is the ability to inject a siRNA into a peripheral vein, and find that the siRNA enters the distant target organ to silence the target mRNA molecule. This target mRNA molecule is hidden behind multiple membrane barriers in vivo that separate the blood compartment from the mRNA. In the present work, the siRNA is mono-biotinylated in parallel with the production of a fusion protein of avidin and a receptor specific monoclonal antibody (MAb). The present work will engineer, express, and validate a new fusion protein comprised of avidin and a mouse/rat chimeric MAb to the mouse transferrin receptor (TfR). The TfRMAb acts as a molecular Trojan horse, to ferry the siRNA across any target cell membrane that expresses the TfR. Since the TfR is highly expressed on the blood-brain barrier (BBB), the availability of the TfRMAb/AV fusion protein will enable in vivo RNAi for the brain, as well as other organs. Eukaryotic expression plasmids encoding the light chain (LC) and the heavy chain (HC)/AV fusion protein will be genetically engineered, expressed in host cells, and the TfRMAb/AV fusion protein will be purified by protein G affinity chromatography, followed by validation of the bi- functionality of the fusion protein with mouse TfR and biotin binding assay. A single tandem vector will be engineered for permanent transfection of host cells. Following selection, amplification, and dilutional cloning, the transfected host cells will be propagated, and the TfRMAb/AV fusion protein will be purified by affinity chromatography. The TfRMAb/AV fusion protein produced at this large scale will be validated with a series of biochemical assays to verify the structural and functional integrity of the molecule, including the validation of siRNA action and RNA interference. PUBLIC HEALTH RELEVANCE RNA interference (RNAi) is a new technology that enables the knockdown of expression of pathologic genes with short interfering RNAs (siRNAs). The limiting factor in the development of new RNAi medicines is delivery. The goal of the field is "Intravenous RNAi," which is the ability to inject a siRNA into a peripheral vein, and find that the siRNA enters the distant target organ to silence the target mRNA molecule. The present work will engineer, express, and validate a new fusion protein comprised of avidin and a mouse/rat chimeric MAb to the mouse transferrin receptor. [unreadable] [unreadable] [unreadable]